Mechanism of recruitment of p53-binding protein 1 (53BP1) to DNA double-strand breaks

p53-binding protein 1 (53BP1) is a conserved nuclear protein that is rapidly recruited to sites of DNA double-strand breaks (DSBs) following DNA damage. Here, we show that the mechanism of recruitment involves recognition of a methylated lysine in histone H3, namely K79, which becomes exposed following break-induced chromatin relaxation. We have solved the crystal structure of the domain necessary for its recruitment, and found that it consists of two tandem Tudor domains with a deep pocket at their interface formed by evolutionarily conserved hydrophobic residues. Single amino acid substitutions of residues that form the walls of the 53BP1 hydrophobic pocket abrogate both binding to methylated histone H3 and also recruitment to sites of DNA DSBs. Second, depletion of Dot1L, the enzyme that methylates histone H3 on Lys79 in vivo, inhibits recruitment of 53BP1 to sites of DNA DSBs. Methylation of histone H3 Lys79 is not enhanced in response to DNA damage, supporting the model that a DNA DSB could lead to disruption of higher order chromatin structure, resulting in nucleosome unstacking and exposure of methylated Lys79 of histone H3. We later demonstrate that the tandem Tudor domain alone is not sufficient for recruitment, and that an upstream oligomerization domain is required for this function. We have mapped the boundaries of this new domain and find it to comprise approximately 40 residues, lying at a distance of around 200 residues upstream of the Tudor. Deletion and point mutations in this domain abolish recruitment to DSBs, but this is rescued by introducing an exogenous oligomerization domain upstream of the Tudor. Further, deletion mutants that are foci-proficient migrate as higher-order oligomers on a gel filtration column, while those that are foci-deficient migrate as monomers. We propose that 53BP1 oligomerization facilitates its interaction with relaxed chromatin that results from DNA breaks